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Chapter 18

Excitatory and Inhibitory SynapticPlacement and Functional Implications

Katherine L. Villa and Elly Nedivi

Abstract Synaptic transmission between neurons is the basic unit of communica-

tion in neural circuits. The relative number and distribution of excitatory and

inhibitory synaptic inputs across individual dendrites and neurons are the hardware

of local dendritic and cellular computations. In this chapter, we discuss the structural

and functional observations that have guided the understanding of excitatory and

inhibitory synaptic organization across the neuronal arbor, the subcellular targeting

properties of different neuronal subtypes, and the effects of synaptic placement on

local integration within dendritic segments. We focus primarily on the adult mam-

malian cortex and hippocampus, where excitatory and inhibitory cell types, their

connectivity, and its functional implications have been best characterized.

Keywords Synaptic placement • Excitatory-inhibitory balance • Dendritic

integration

18.1 Introduction

The neurotransmitter synthesized and released at the synapse is the basis for the

classification of neurons as excitatory or inhibitory. Glutamatergic neurons, releas-

ing glutamate, excite postsynaptic cells, while GABAergic neurons, releasing

GABA (γ-aminobutyric acid), inhibit them. The delicate balance between

K.L. Villa

Picower Institute for Learning and Memory, Massachusetts Institute of Technology,

Cambridge, MA 02139, USA

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

E. Nedivi (*)

Picower Institute for Learning and Memory, Massachusetts Institute of Technology,


Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology,


e-mail: [email protected]

© Springer Japan 2016

K. Emoto et al. (eds.), Dendrites, DOI 10.1007/978-4-431-56050-0_18467

mailto:[email protected]

excitation and inhibition is essential for precise nervous system function and

plasticity, as its perturbation has been associated with disorders ranging from

epilepsy (Mohler et al. 2004), to autism (Rubenstein and Merzenich 2003), and

mental retardation (Dani et al. 2005; Kleschevnikov et al. 2004), as well as

neuropsychiatric disorders such as schizophrenia (Lewis et al. 2005). A key to

this balance is the precise coordination of excitatory and inhibitory synaptic activity

at specific cellular locales. Yet, our knowledge regarding global distribution, as

well as the local placement, of excitatory and inhibitory synaptic inputs across

different neuronal subtypes is still quite poor. Below we summarize the current

state of the field and the evolution of our understanding to date.

18.2 Organization of Synapses Across the Dendritic Arbor

18.2.1 First Views Through Golgi Stainingand Electron Microscopy

18.2.1.1 Pyramidal Neurons

In the late nineteenth century, development of the Golgi stain, a silver stain that

sparsely but intensely labels neurons, enabled the first clear discrimination of

individual neuronal morphology (Fig. 18.1A–C). In his meticulously detailed

drawings, Ramon y Cajal characterized the elaborate dendritic arbors and axonal

Fig. 18.1 Visualization of L2/3 pyramidal neurons. (A) Golgi-stained pyramidal neuron (Image

kindly provided by Dr. Terry Robinson, University of Michigan). (B,C) High-magnification views

of the two dendrites marked by arrows in panel A. Note dendritic spines. (D) 3D volume

projection of a pseudo-colored pyramidal neuron imaged in vivo using two-photon microscopy.

(E) An individual dendrite expressing YFP (red) and teal-gephyrin (green) to label inhibitory

synapses. (F) The same dendrite imaged 8 days later. Yellow arrows indicate dynamic inhibitory

synapses and red arrows indicate dynamic spines. Filled arrows show when structures are present

and open arrows indicate their absence

468 K.L. Villa and E. Nedivi

processes of neurons with a wide range of morphologies. One obvious distinction

was that some neurons have smooth dendrites, while others have many spiny

protrusions. In general, most neurons with spiny dendrites were later revealed to

be glutamatergic and excitatory, while neurons with smooth dendrites for the most

part release GABA and are inhibitory (Gabbott and Somogyi 1986; Kubota 2014;

Morishima et al. 2011; Thomson and Deuchars 1997). Elegant electron microscopy

(EM) studies on Golgi-stained cells observed that each spine contains a synapse

characterized by round presynaptic vesicles and a robust postsynaptic density

(Hersch and White 1981; LeVay 1973; Parnavelas et al. 1977). These asymmetric

synapses, classified as type 1 synapses, are innervated by axons of glutamatergic

neurons (Baude et al. 1993). While there are a few reports of specific cell regions on

particular types of neurons where type 1 synapses are localized directly on the

dendritic shaft (Megias et al. 2001; Parnavelas et al. 1977), this is generally not the

case. Given that the vast majority of spines, with the exception of some very thin

spines (about 2–4% of total cortical spines) (Arellano et al. 2007; Hersch and

White 1981; White and Rock 1980), have a single type 1 excitatory synapse

(Harris et al. 1992; LeVay 1973), it was reasonable to assume that each spine can

serve as a readily identifiable structural surrogate for an excitatory synapse. Thus,

for spiny pyramidal neurons, dendritic spine distributions as seen by Golgi stain or

by filling cells with a fluorescent dye could inform as to the placement of

excitatory synapses across the dendritic arbor (Elston and Rosa 1997; Larkman

1991; Trommald et al. 1995).

There is a great variety in the density of spines between cells and between

different branches on the same cell. To give a general sense of spine distributions,

spine density is low within 40 μm of the cell body, reaching a maximum density in a

region 40–130 μm from the soma, and then gradually decreasing toward a den-

drite’s distal tips (Megias et al. 2001). On the distal branches of pyramidal cells,

spine density can range from 0 to 70 spines per 10 μm (Megias et al. 2001) with the

highest spine density often found on the thickest dendrites, usually the primary

apical dendrite. The total number of spines on spiny excitatory neurons typically

ranges from 5,000 to 35,000 spines per cell, ultimately depending on the total

dendritic length, with L5 pyramidal cells generally having longer total dendrite

length and therefore more synapses than L2/3 pyramidal cells (Larkman 1991).

Inhibitory synapses innervated by the axons of GABAergic neurons, classified

by EM as type 2, or symmetric synapses, are typified by a symmetric synaptic cleft,

due to a minimal postsynaptic density, as well as flattened presynaptic vesicles

(Davis and Sterling 1979). Unfortunately, since type 2 inhibitory synapses lack a

morphological surrogate, their distribution patterns cannot be discriminated by

Golgi staining alone. Laborious EM reconstructions of relatively small neuronal

regions have shown excitatory to inhibitory synaptic ratios along pyramidal den-

drites ranging from 6.5–12.5 to 1 (Davis and Sterling 1979). As previously men-

tioned, the cell body and proximal dendrites of excitatory neurons typically lack

spines. However, they are often densely innervated by type 2 synapses. In the

aspiny region of the proximal dendrites, inhibitory synapse density can be as high as

17 synapses per 10 μm (Megias et al. 2001). EM studies also show that on

18 Excitatory and Inhibitory Synaptic Placement and Functional Implications 469

excitatory dendrites, the majority of type 2 inhibitory synapses are located on the

dendritic shaft at a density of about 3 per 10 μm (Hersch and White 1981).

Inhibitory synapses can also be located on dendritic spines adjacent to excitatory

synapses (Parnavelas et al. 1977), as well as on the axon initial segment (Hersch and

White 1981; Westrum 1966). Inhibitory axons from different inhibitory neuron

subtypes specifically target discrete regions of their postsynaptic pyramidal cell

partners (we discuss this in greater detail further in the chapter).

18.2.1.2 Non-pyramidal Neurons

The dendrites of inhibitory neurons, in general, do not contain spines. However, a

small subset of inhibitory neurons have dendritic spines (Azouz et al. 1997; Feldman

and Peters 1978), with densities that range from 0.3 to 7 spines per 10 μm (Kawaguchi

et al. 2006; Keck et al. 2011). Like spiny pyramidal cells, the cell body and most

proximal dendrites of spiny interneurons within 30 μm of the soma lack spines

(Kawaguchi et al. 2006). There are several distinct subtypes of spiny inhibitory

neurons, but Martinotti cells are the ones with the highest spine density with about

three to seven spines per 10 μm, approximately one fourth of the density on pyramidal

cells (Gulyas et al. 1992; Kawaguchi et al. 2006). Martinotti cells also have longer

spines than other spiny interneuron subtypes andmoremultiheaded spines (Kawaguchi

et al. 2006). Immunohistochemistry experiments show that the majority of spines on

these interneurons colocalize with Vglut1 but not VGAT, indicating that they mostly

harbor excitatory synapses (Keck et al. 2011). Unlike dendritic spines on excitatory

neurons, which typically contain only one excitatory synapse, the spines of some spiny

inhibitory neurons in the hippocampus can contain up to six distinct excitatory

synapses (Gulyas et al. 1992). The proportion of excitatory synapses located along

the shaft of spiny inhibitory dendrites has not been established. We also know

little about the distribution of inhibitory synapses on spiny interneurons (Gulyas

et al. 1992).

Aspiny interneurons receive both excitatory and inhibitory inputs onto their

soma and proximal dendrites (Davis and Sterling 1979), with a higher density

along their distal dendrites (Parnavelas et al. 1977). Similarly to inhibitory synapses

on pyramidal cells, both excitatory synapses and inhibitory synapses onto dendrites

of aspiny inhibitory neurons cannot be visualized through a morphological surro-

gate and could initially be examined only by EM on relatively small arbor segments

or by immunohistochemistry. EM reconstructions of isolated branches from differ-

ent inhibitory neuron types provide anecdotal evidence that parvalbumin (PV)-

positive cells have the highest density of synapses with a density of 30 excitatory

and 2 inhibitory synapses per 10 μm of dendritic length for branches sampled

(Gulyas et al. 1999). Inhibitory synapses are also relatively rare on calretinin

(CR)-positive spiny interneurons (Gulyas et al. 1992) which have a lower density

of seven excitatory synapses and two inhibitory synapses per 10 μm (Gulyas

et al. 1999).


18.2.2 Synaptic Visualization by New In Vivo FluorescentLabeling Methods

Golgi staining gave us a first view of dendritic spine distributions, and EM studies

were first to shed light on the fundamental layout of excitatory and inhibitory

synaptic distributions on different neuronal types. Yet, both methods have inherent

limitations. As a cell fill, Golgi stain can at best identify spines on cells with

spiney dendrites, while all inhibitory synapses as well as excitatory synapses on

aspiny dendrites remain invisible. EM is limited by the difficulty of reconstructing

large dendritic segments. EM reconstruction of an entire cell would require a

heroic effort, one rarely attempted (see White and Rock 1980 for a reconstruction

of an entire spiny stellate neuron). Both Golgi staining and EM reconstructions

necessitate tissue fixation and cannot be used for visualizing structural dynamics.

Thus, our initial view of synaptic distributions was constrained by the limitations of

the Golgi and EM methods.

Recently, imaging of fluorescently labeled cells in vivo has provided a new view

not only of dendritic spine distributions but also of excitatory and inhibitory

synapses as well as the dynamics of these structures (Fig. 18.1D–F). First studies

on neurons fluorescently labeled with green fluorescent protein (GFP) showed that

in adult animals dendrites of pyramidal neurons are very stable (Grutzendler

et al. 2002; Mizrahi and Katz 2003; Trachtenberg et al. 2002), but dendritic spines

are highly dynamic (Trachtenberg et al. 2002), implying a capacity for synaptic

removal and addition. Spine dynamics of excitatory as well as spiny inhibitory

neurons can be further increased upon sensory deprivation (Hofer et al. 2009;

Holtmaat et al. 2006; Keck et al. 2008; Trachtenberg et al. 2002). Dynamics differ

dependent on deprivation protocol and cortical lamina, consistent with the view that

they reflect specific circuit alterations.

In contrast to the stability of excitatory dendritic branches, inhibitory dendrites

are capable of growth and retraction in vivo (Chen et al. 2011a, b; Lee et al. 2006,

2008), and their dynamics are influenced by sensory manipulations (Chen

et al. 2011b). The boutons of inhibitory neurons are also capable of remodeling,

and their dynamics also increase in response to sensory deprivation (Chen

et al. 2011b; Keck et al. 2011; Pieraut et al. 2014; Schuemann et al. 2013; Wierenga

et al. 2008). Thus, synaptic distributions are not necessarily rigid. Rather, both

excitatory and inhibitory synapses are dynamic structures, and their remodeling

potentially underlies functional plasticity.

More recently, the expression of fluorescent proteins fused to postsynaptic

scaffolding molecules has enabled direct synaptic visualization in vivo. Expression

of fluorescent proteins fused either to PSD95, as a postsynaptic marker of excitatory

synapses, or gephyrin, as a postsynaptic marker of inhibitory synapses, together

with a separate cell fill to outline their location on the arbor has allowed for the

first time a direct assessment of synaptic distribution patterns on cortical pyramidal

cells, as well as synapse dynamics at different cellular locales (Cane et al. 2014;

Chen et al. 2012; Isshiki et al. 2014; van Versendaal et al. 2012; Villa et al. 2016).


A critical aspect of this approach is the sparse labeling of only a subset of neurons

so that accurate counting of synaptic distributions is feasible, in a way that is not

possible with antibody staining. PSD95-GFP labeling in vivo shows that

most stable spines have large PSD95 puncta, but a small population are devoid of

a PSD95 label (Cane et al. 2014; Villa et al. 2016). While these spines

may contain other PSD95 scaffolding molecules, such as PSD93 or SAP

102 typical of immature synapses (Aoki et al. 2001; Elias et al. 2008; Sans

et al. 2000), this finding highlights the fact that spines are an imperfect surrogate

for excitatory synapses.

When labeling postsynaptic gephyrin as a marker for inhibitory synapses on

pyramidal neurons, a surprising finding was the prevalence of inhibitory synapses

on dendritic spines (Chen et al. 2012; van Versendaal et al. 2012). Dually inner-

vated spines harboring both an excitatory and an inhibitory synapse had previously

been reported by EM (Fifkova et al. 1992; Kisvarday et al. 1985; Knott et al. 2002;

Kubota et al. 2007; Megias et al. 2001; Parnavelas et al. 1977). However, the

fluorescent labeling provided an orders of magnitude larger sample size as com-

pared to EM, leading to the realization that on average, a third of inhibitory

synapses on L2/3 pyramidal neurons reside on spines. Large volume imaging of

entire L2/3 pyramidal neurons, labeled with fluorescent gephyrin in addition to a

cell fill, allowed a first quantitative look at inhibitory synapse distributions across

the dendritic arbors of this cell type: on average five spines and two inhibitory

synapses per 10 μm (Chen et al. 2012). About 15% of spines contain an inhibitory

synapse, with an overall density of one inhibitory synapse on a spine per 10 μm(Chen et al. 2012). Both spines and inhibitory shaft synapses are distributed

relatively uniformly across the spiny region of the dendritic arbor. In contrast,

inhibitory spine synapses were found to be denser on the apical tuft than on basal

and proximal dendrites (Chen et al. 2012).

18.3 Segregation of Distinct Inputs onto Specific CellularLocales

18.3.1 Interneuron Targeting of Pyramidal Neurons

While synaptic distributions and the valance of the input at each location are

important aspects of an individual neuron’s wiring diagram, identifying the precise

source of input at each synaptic locale is also critical for understanding a neuron’scomputational properties. GABAergic cells are particularly heterogeneous with

respect to their axonal and dendrite morphology, firing patterns, molecular markers,

and postsynaptic targeting (Klausberger and Somogyi 2008) and can be classified

into subtypes based on these features (DeFelipe et al. 2013; Petilla Interneuron

Nomenclature et al. 2008) or by staining for molecular markers, such as

parvalbumin (PV), somatostatin (SOM), the serotonin 5HT3a receptor (5HT3aR),


vasoactive intestinal peptide (VIP), calbindin D (CB), and CR (Kawaguchi and

Kubota 1997; Kubota et al. 1994, 2011). The PV, SOM, and 5HT3aR markers

define nonoverlapping classes of neurons and together represent nearly 100% of

cortical interneurons (Lee et al. 2010; Rudy et al. 2011; Uematsu et al. 2008).

The majority of inhibitory neurons expressing the calcium-binding protein PV,

classified as fast-spiking basket neurons, predominantly form synapses onto the

soma and proximal dendrites of excitatory neurons (Miles et al. 1996) (Fig. 18.2a).

In the G42 transgenic line, which labels a subset (~50%) of PV-positive cells

(Chattopadhyaya et al. 2004), 20% of synapses onto pyramidal cells target the

soma and 40% the proximal dendrites (within 40 μm of the soma) (Di Cristo

et al. 2004). The remaining 40% target the more distal pyramidal neuron dendrites

(Di Cristo et al. 2004). Thus, although PV cells are more likely to innervate the

soma and proximal dendrites of pyramidal neurons, they are not strictly limited to

these subcellular domains. PV cells strongly inhibit neighboring pyramidal neurons

(60% probability of connectivity) (Avermann et al. 2012; Lee et al. 2013). Because

of the large number of PV cell inputs targeting the soma and proximal dendrites of

pyramidal neurons, PV neurons are capable of regulating the timing and magnitude

of pyramidal output and can limit the temporal window of action potential gener-

ation (Higley 2014; Pouille and Scanziani 2001).

Fig. 18.2 Schematic circuit diagrams of cortical neurons illustrating general principles of con-

nectivity. (A) Schematic of inhibitory innervation of a cortical pyramidal neuron (red) by cell

types that target distinct cellular locations. Chandelier cells (green) target the axon initial segment.

Parvalbumin (PV)-expressing interneurons (orange) mostly target the soma and proximal den-

dritic segments. Somatostatin (SOM)-expressing interneurons (blue) mostly target distal dendrites.

(B) Schematic of how interneurons target other interneurons. In addition to pyramidal neurons, PV

cells (orange) mostly innervate other PV cells. VIP-positive interneurons (purple) primarily target

other interneurons and rarely innervate pyramidal neurons. In L2/3, SOM interneurons (blue)mostly target pyramidal neurons and are less likely to innervate other interneurons. (C) Schematic

of excitatory cortical circuitry. Thalamic axons (orange) drive activity of L4 spiny stellate neurons(light blue) and innervate L1 apical tufts. Stellate neurons innervate the proximal dendrites of L2/3

neurons (pink) which in turn project distantly to other cortical regions and to the proximal

dendrites of nearby L5 and L6 pyramidal neurons (red and purple), which project to subcortical

and thalamic regions, respectively. Top-down cortical inputs (blue) primarily innervate L1 apical

tufts


Chandelier cells, or axo-axonic cells, are a morphologically distinct GABAergic

cell type recognizable by their axonal arborizations. These cells innervate the axon

initial segment of neighboring pyramidal cells (Fig. 18.2a), giving their own axonal

arbor a stereotypical chandelier-like appearance (Somogyi 1977; Somogyi

et al. 1982; Woodruff et al. 2010, 2011). A widely held notion is that chandelier

cells are a subset of PV-positive interneurons, but recent work shows that only a

subset of chandelier cells stain positive for PV (Kawaguchi and Kubota 1998;

Taniguchi et al. 2013). Through their innervation of the axon initial segment,

chandelier cells can be seen as the last line of defense for preventing the post-

synaptic cell from firing an action potential, although they can also have

depolarizing effects, depending on the membrane potential of the postsynaptic

neuron (Khirug et al. 2008; Szabadics et al. 2006; Woodruff et al. 2009).

SOM interneurons expressing the peptide hormone somatostatin typically target

the dendrites of pyramidal neurons (Kawaguchi and Kubota 1998) (Fig. 18.2a).

One well-known SOM cell subtype, the Martinotti cell, has a clearly identifiable

morphology, with an axon that projects apically to layer 1 and there inhibits the

apical tufts of deep-layer pyramidal cells. Although SOM-positive neurons are

more inclined to innervate the most distal dendrites (Jiang et al. 2013), it is

inaccurate to categorize their synapses as exclusive to that cellular locale. In the

GIN transgenic line which labels a subset (~20%) of SOM-positive cells (Oliva

et al. 2000), pyramidal cell targeting by these neurons is such that 4% of their

synapses are directly onto the soma, 20% are onto the proximal dendrites (within

40 μm of the soma), and the remaining 76% are onto the more distal dendrites

(Di Cristo et al. 2004). SOM interneurons are highly likely to innervate neighboring

pyramidal cells (71% probability of innervating cells within 200 μm) and also

target other interneuron subtypes, but specifically avoid innervation of other

SOM-positive neurons (Fig. 18.2b) (Fino and Yuste 2011; Pfeffer et al. 2013). By

innervating the dendrites of pyramidal cells, SOM interneurons are likely to play an

important role in controlling the generation of dendritic Ca2+ spikes (discussed

later), and they can deliver more localized control over particular excitatory inputs.

They are positioned to modulate the excitability of individual dendritic spines,

segments, or branches.

18.3.2 Interneuron Targeting of Other Interneurons

The different subsets of interneurons also have specific targeting patterns onto other

interneurons (Fig. 18.2b). PV cells have also been shown to strongly inhibit

neighboring PV cells (with 55% probability of connectivity), but are less likely

to innervate other interneuron subtypes (24% probability of connectivity with

neighboring 5HT3aR neurons) (Avermann et al. 2012; Pfeffer et al. 2013).

5HT3aR-positive neurons have only been recently described and can be divided

into two distinct subgroups. The first is VIP negative and typically reelin positive,

which has a late-spiking firing pattern and a neurogliaform axonal arbor


morphology restricted to L1 (Lee et al. 2010; Rudy et al. 2011). The second is VIP

positive and represents about 15% of all GABAergic cells. These neurons mostly

target SOM and some PV interneurons, and thus their firing results in disinhibition

of pyramidal cells (Hioki et al. 2013; Pfeffer et al. 2013; Pi et al. 2013).

VIP-positive interneurons are highly unlikely to innervate neighboring pyramidal

cells. Paired recordings show that only 12% of VIP neurons innervate a pyramidal

neighbor (Pfeffer et al. 2013). In a recently described mouse circuit, pyramidal

neurons within vibrissal motor cortex (vM1) that project to somatosensory barrel

cortex (S1) strongly innervate VIP neurons, which in turn innervate SOM neurons

that target S1 pyramidal neurons (Lee et al. 2013). This circuit activates S1 VIP

neurons during whisking, resulting in the inhibition of S1 SOM neurons and thus

disinhibition of S1 pyramidal neurons, potentially influencing the coincident detec-

tion of sensory information and altering whisker movement (Lee et al. 2013).

More generally, through their inhibitory control over other inhibitory circuits and

their location in superficial cortical layers, VIP cells likely play important roles in

regulating inter-areal cross-talk between different cortical regions.

Some SOM neurons in L4 of the somatosensory cortex have also been shown to

target other GABAergic interneurons, so that their activation results in a dis-

inhibition of pyramidal cells (Jiang et al. 2013; Xu et al. 2013). The SOM neurons

in layer 4 differ in morphology, intrinsic electrophysiological properties, and output

connectivity from L2/3 SOM interneurons. Specifically, L2/3 SOM interneurons

make many strong contacts with pyramidal neurons and suppress pyramidal activ-

ity, while L4 SOM neurons make frequent strong contacts with PV-positive

fast-spiking interneurons, which themselves inhibit L4 pyramidal neurons

(Xu et al. 2013). Thus, increasing the activity of L4 SOM neurons results in

increased activity of L4 pyramidal neurons (Xu et al. 2013). These L4 SOM

neurons are not strongly activated by thalamic inputs, but are likely activated by

acetylcholine (Fanselow et al. 2008; Kawaguchi 1997), which suggests that during

arousal and attention, L4 SOM neurons can become activated, resulting in greater

activation of L4 pyramidal neurons.

18.3.3 Pyramidal Cell Targeting of Other PyramidalNeurons

Is there a subcellular bias to excitatory axon innervation of cortical excitatory

targets? Historically, unlike inhibitory neurons, excitatory pyramidal cells have

been considered a generally uniform population, with circuit specificity resulting

from their laminar location. Recent molecular studies have identified genes that are

expressed in specific cortical layers (Belgard et al. 2011; Molyneaux et al. 2007), as

well as genetic markers resolving intra-layer subsets of excitatory neurons distin-

guished by their projections to specific target structures (Sorensen et al. 2015).

When excitatory neurons are defined according to their axonal projection and target


innervation, pyramidal neuron diversity may exceed that of inhibitory neurons

(Huang 2014). Potentially, resolving excitatory neuron subclasses may also reveal

distinct subcellular target specificity analogous to that of interneuron subclasses. In

the hippocampus, considered a simplified cortical structure containing only one

pyramidal cell layer (Thomson and Lamy 2007), genetically distinguishable pyra-

midal neuron subsets have not yet been identified. Cre recombinase driver lines

created by the GENSAT project should prove useful for further resolution of

pyramidal neuron subtypes (Gerfen et al. 2013; Gong et al. 2007).

Within the cortex, pyramidal cell dendrites can receive excitatory inputs from

several sources: from local axons within the same cortical column, from adjacent

cortical column axons, from other cortical regions in the same hemisphere, from

commissural axons originating in the opposite hemisphere, as well as from direct

feed-forward inputs that originate from the thalamus (Fig. 18.2c). The main target

of feed-forward thalamic axons is L4, and indeed L4 cells are strongly activated by

the thalamus despite the fact that cortical synapses onto L4 outnumber thalamic

synapses by a factor of 10 (Benshalom and White 1986; Peters and Payne 1993).

Thalamic synapses onto L4 are localized slightly more proximal to the soma than

corticocortical synapses, but their synaptic strengths are approximately equal,

suggesting that coincident activation of thalamic synapses is the main contributing

factor to the strength of thalamocortical synapses (Schoonover et al. 2014). The

classic thalamic information flow through the cortex is considered to pass from L4

to L2/3, then down to L5 and L6, and out to other cortical areas and back down to

the thalamus and other subcortical regions. However, L2/3, L5, and L6 receive

sparse thalamic inputs as well (for excellent reviews of cortical innervation patterns

in the barrel cortex, see (Feldmeyer 2012; Lefort et al. 2009)), and recent in vivo

studies show that thalamic inputs can drive L5/L6 independently of L4 (Constanti-

nople and Bruno 2013).

Given the laminar nature of information flow within the cortex, the relative

placement of a dendritic or other cellular locale within the cortical lamina is the

strongest determinant of excitatory input specificity onto that locale. Apical den-

drites of L2/3, L5, and L6 pyramidal cells arborizing in L1 receive feedback

excitatory inputs from other cortical areas as well as from the posterior medial

thalamic nucleus (POm) axons that travel in this lamina (Felleman and Van Essen

1991; Larkum 2013). Local, interlaminar cortical axons carrying feed-forward

information are more likely to innervate the basal and proximal dendrites of their

cortical targets (Feldmeyer 2012; Spruston 2008). For example, L4 axons mainly

innervate the basal dendrites and proximal apical dendrites of L2/3 pyramidal cells

within 70 μm of the soma (Feldmeyer et al. 2002; Silver et al. 2003), and L2/3

pyramids predominantly innervate the basal dendrites of L5 pyramidal neurons

(Schubert et al. 2001). There is also a bias for thalamic axons to innervate the basal

dendrites of L5 pyramidal neurons (Feldmeyer 2012). On the other hand,

corticocallosally projecting L5 pyramidal neurons synapse mainly onto the distal

region of the basal dendritic tree of their L5 partners at an average distance of about

130 μm (Larsen and Callaway 2006), suggesting that commissural axons are more

likely to innervate the distal dendrites of their targets. There are also examples of


subcellular innervation bias in the hippocampus, where the CA1 pyramidal cells are

innervated by axons of CA3 and entorhinal cortex (EC); the EC axons preferentially

target the distal dendrites of CA1 neurons, and the CA3 axons innervate the basal

and proximal dendrites of the same CA1 neurons (Megias et al. 2001). Additionally,

distant CA3 neurons are more likely to project to the apical CA1 dendrites, while

CA3 neurons with cell bodies located close to CA1 are more likely to project to the

basal dendrites of CA1 neurons (Li et al. 1994). Thus, a general theme seems to be

that long-range feedback excitatory inputs are more likely to innervate distal

dendrites, while feed-forward thalamic and interlaminar projections are more likely

to target basal and proximal dendrites of their cortical targets. Although we have yet

to delineate the full extent of cellular and subcellular targeting specificity for each

cortical cell type, the precision we see thus far suggests that excitatory and

inhibitory synaptic placement onto pyramidal cell dendrites is a critical aspect of

their function within the circuit.

18.4 Functional Implications of Synaptic Placement Acrossthe Dendritic Arbor

18.4.1 Spatial Clustering of Excitatory Inputs

Experiments suggest that neurons are capable of three different types of dendritic

integration. Inputs can be summed sublinearly, when there is a decrease in driving

force as the membrane potential nears the reversal potential; linearly, when inputs

occur on different dendritic branches or are separated by sufficient distance to act

independently; and supralinearly, when local conditions facilitate depolarization

(Palmer 2014). These different modes of processing are possible within the same

neuron, for example, if the dendrites operate linearly when activated by a

non-preferred stimulus and nonlinearly when activated by a preferred stimulus

(Grienberger et al. 2015). The type of integration performed is strongly influenced

by the relative placement of excitatory and inhibitory synapses on the dendrite as

well as the dendrite’s location within the arbor.

Historically, synapses distant from the soma were thought to have less influence

over action potential initiation in the axon, due to loss of charge as current flows

from the dendrites to the soma and axon hillock in a model of passive dendrites

(Golding et al. 2005). However, the discovery of dendritic spikes explained how

distal excitatory synapses are able to propagate their signals effectively to the soma.

Dendritic spikes are essentially action potentials that are generated in the dendrite,

which acts as an active rather than passive signaling compartment. As dendritic

spikes propagate toward the soma, they are capable of triggering action potentials

(Golding and Spruston 1998), also recently shown in vivo (Grienberger et al. 2014).

Dendritic Ca2+ spikes can be initiated by strong synchronous activation of several

synapses (Gasparini and Magee 2006), and the probability of dendritic spike


initiation in distal branches can be further increased by nearby initiation of NMDA

spikes (Larkum et al. 2009). Theoretical models and in vitro experiments both

support the view that spatially clustered, temporally coactivated excitatory inputs

are more likely to initiate dendritic spikes than inputs that are distributed across

multiple branches (Gasparini and Magee 2006; Larkum et al. 2009; Mel 1993;

Winnubst and Lohmann 2012). Dendritic spikes can thus improve the computa-

tional properties of individual neurons (Larkum 2013; Major et al. 2013), by

enabling the discrimination of patterns delivered to a single dendrite from those

delivered randomly across the dendritic tree (Branco et al. 2010).

Another consequence of the fact that dendritic spikes elicited by nearby coactive

synapses are capable of triggering action potential firing is that neighboring syn-

apses would thus be more likely to undergo long-term potentiation (LTP) than

synapses that are spatially distant. There are also examples of dendritic spike-

induced LTP independent of action potential firing in vitro (Golding et al. 2002;

Gordon et al. 2006) and recently in vivo (Gambino et al. 2014). Since dendritic

spikes have a smaller spread than action potentials, these would preferentially

produce LTP on locally clustered coactive synapses, especially in regions of the

distal dendritic tree that are beyond the reach of backpropagating action potentials

(Golding et al. 2001). The pervasiveness of functionally clustered inputs in vivo is

under debate. Studies in the visual (Jia et al. 2010), auditory (Chen et al. 2011c), and

barrel cortex (Varga et al. 2011) have suggested that functionally distinct excitatory

inputs are randomly distributed along pyramidal dendrites. Other experiments

provide evidence for clustering of functionally related excitatory inputs in the

hippocampus (Druckmann et al. 2014; Kleindienst et al. 2011; Takahashi

et al. 2012) and cortex (Chen et al. 2013; Makino and Malinow 2011; McBride

et al. 2008; Takahashi et al. 2012) (reviewed in DeBello et al. 2014). These findings

are not necessarily in conflict. Excitatory synapses that are functionally similar may

have a higher probability of spatial clustering along the dendrite, but at the same

time, dendrites are interspersed with a wide range of functional inputs. Spatial

clustering of functionally related inputs may also be more prominent in certain

subsets of neurons.

18.4.2 Effects of Inhibitory Synaptic Placementon Pyramidal Cell Dendritic Integration

Individual pyramidal cells receive thousands of excitatory synaptic inputs that can

be functionally diverse. Yet, the action potential output of most pyramidal neurons

is sparse and narrowly tuned. This restriction of action potential firing to a defined

sensory range within a limited temporal window is brought about by the integration

of excitatory synaptic events combined with the local influence of inhibition

(reviewed in Chadderton et al. 2014). Inhibition has a major influence on the

effective output of neurons at multiple levels. Individual inhibitory synapses on


spines can veto individual spine conductances (Chiu et al. 2013). Inhibitory shaft

synapses can influence excitatory integration within local dendritic segments (Kim

et al. 1995; Perez-Garci et al. 2006; Willadt et al. 2013). On a more cellular scale,

PV cell innervation of the soma (Pouille and Scanziani 2001) or chandelier cell

innervation of the axon initial segment (Woodruff et al. 2011) can cancel axonal

output. In the latter cases, the high density of inhibitory synapses on the cell body

and axon initial segment (Di Cristo et al. 2004; Miles et al. 1996; Woodruff

et al. 2010) suggests that synchronous firing of many synapses would be needed

for effective inhibition in these locales, and a single inhibitory synapse would have

a negligible effect on outcome. This is in contrast to dendritically targeted inhibi-

tion, where individual synapse changes could have a dramatic effect on the com-

putation of dendritic integration for specific branches.

How are excitatory and inhibitory inputs integrated functionally at the level of

individual dendritic branches and how does that influence overall neuronal firing?

Clearly, local integration of excitatory and inhibitory synaptic activity is influenced

by individual synaptic strength, timing, and location on the dendrite. Theoretical

modeling predicts that activation of multiple distributed inhibitory synapses across

the dendritic arbor can result in a global inhibition (Gidon and Segev 2012).

However, experiments suggest that the influence of individual inhibitory synapses

is locally restricted to the branch they target (Stokes et al. 2014), perhaps even to

individual spines (Chiu et al. 2013). Thus, functional pairing or clustering of

relevant excitatory synapses on particular dendritic segments with their inhibitory

neighbors is highly relevant to dendritic integration. Moreover, the local constella-

tion of such pairing or clustering is likely to have a different influence depending on

their more global location along the arbor.

Dendritically targeted inhibition can limit initiation of dendritic Ca2+ spikes in

both hippocampal and cortical pyramidal neurons (Buhl et al. 1994; Karube

et al. 2004; Kim et al. 1995; Larkum et al. 1999; Perez-Garci et al. 2006; Willadt

et al. 2013), so that they are initiated only in response to inputs encoding particular

functional features. For example, in retinal ganglion cells, local integration of

excitation and inhibition leads to initiation of dendritic spikes only in the preferred

direction of motion, with inhibition preventing dendritic spike initiation in the

non-preferred direction (Sivyer andWilliams 2013). Mathematical models focusing

on spike activity within dendrites have shown that “off path” inhibitory synapses,

where the inhibitory synapse is located distally to the excitatory synapse, are more

effective at preventing the initiation of dendritic spikes (Gidon and Segev 2012).

Thus, the influence of inhibition on spike initiation can actually be more powerful

when inhibitory synapses are located on distal dendrites rather than proximal

dendrites and in a constellation where the inhibitory synapse is distal to its func-

tionally paired excitatory synapse (Gidon and Segev 2012; Li et al. 2014). Con-

versely, in cases where a dendritic spike is successfully initiated, an “on path”

inhibitory synapse, located between the excitatory synapse and the soma, is the

most effective way to dampen the signal (Gidon and Segev 2012; Hao et al. 2009;

Zhang et al. 2013).


Recent in vivo work has demonstrated that dynamic inhibitory synapses are

likely to be located within 10 μm of dynamic spines, and this clustering of dynamic

events increases upon sensory deprivation (Chen et al. 2012). It has been suggested

that a loss of inhibition creates a permissive environment for excitatory synaptic

changes (Chen et al. 2011b; Harauzov et al. 2010; Maya Vetencourt et al. 2008)

because it broadens the window of spike-timing-dependent plasticity (Bi and Poo

2001; Higley and Contreras 2006; Song et al. 2000; Spruston 2008). The loss of an

inhibitory synapse could create a permissive environment for neighboring excit-

atory synaptic gain or loss, while the addition of an inhibitory synapse could

suppress plasticity in neighboring dendritic regions. Inhibitory inputs could also

limit the ability to promote excitatory synaptic clustering through plasticity mech-

anisms. A recent study using calcium imaging in slice culture showed that GABA

uncaging diminishes the Ca2+ transient resulting from a backpropagating action

potential 20 μm in either direction of the uncaging site (Hayama et al. 2013). Thus,

the specific placement of inhibitory synapses can regulate which excitatory synap-

ses are able to undergo plasticity. Further, a recent modeling study suggests that the

placement of inhibitory synapses can result in LTP, LTD, or a lack of plasticity in

specific neighboring dendritic segments (Bar-Ilan et al. 2012), highlighting the

importance on the placement of inhibitory synapses on the valance of plasticity at

neighboring excitatory synapses.

18.5 Conclusion

How the excitatory and inhibitory inputs are integrated within a single neuron and

how this integration supports computation in functioning networks are still critical

questions in neuroscience today. Despite advances in our knowledge, we still have

limited experimental evidence of the fine-scale synaptic architecture across the

neuronal arbor of individual cells of different types, and we know even less of the

functional interactions between synaptic excitation and inhibition (Chadderton

et al. 2014). As we look to the future, combining structural two-photon imaging

of synaptic locations with calcium functional imaging or electrophysiology pro-

vides a potential avenue to address these questions, in cell culture, in acute brain

slices, and ideally in an intact in vivo system.

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